Part 1The first part of the research plan is to assess highly attenuated recombinant poxvirus vaccine candidates in the rhesus macaque model. This work is a collaborative effort with Pasteur Merieux Connaught, which has a CRADA with the NCI, and involves intra-agency collaboration with the NIAID. Two vectors are being tested: a canarypox-based vector (ALVAC) and the genetically attenuated vaccinia strain (a derivative of the Copenhagen strain) NYVAC. Both vectors are able to complete their replication cycle and express heterologous genes under the control of early or early/late promoters. The working hypothesis is that, because both poxvirus vectors, even in the absence of a complete life cycle, still express a large number of their proteins, the response against the heterologous antigens may be limited. Thus we are investigating the hypothesis that conventional DNA plasmid (optimized for expression) or DNA replication (based on the Semliky forest virus replicon) may prime a sufficient immune response that may then be amplified by further boosting with recombinant poxviruses carrying the same SIV genes. Furthermore, we are also testing the importance of the addition of all the accessory/regulatory genes to the SIV/HIV structural genes in these vaccine modalities. Once an optimal combination of modalities is achieved, other regimens of immunization may be further optimized by investigating the importance of the route of immunization and the help of immunomodulating molecules. Part 2In view of the encouraging results obtained with a preventive NYVAC-SIV-gag-pol-env vaccine candidate in rhesus macaques (1), we hypothesized that such a vaccine could also be effective in a regimen of immune intervention following SIV infection. Thus, we develop a model in macaques whereby to test the effect of vaccination in the presence or absence of antiretroviral therapy. The study was designed to assess the following:(1) whether prolonged antiretroviral therapy had any effect on viral rebound following therapy suspension, (2) whether vaccination of macaques could enhance SIV-specific CD4+ T-helper and CD8+ T-cell cytotoxic responses, and (3) whether vaccination increased the ability of the host to maintain viremia at low levels following antiretroviral therapy suspension. The highly attenuated poxvirus NYVAC-SIV-gag-pol-env live recombinant vaccine candidate was chosen as a vaccine approach to test these concepts because of its demonstrated effectiveness as a preventive vaccine candidate in macaques. Three groups of eight macaques were used in the study, each group containing three Mamu-A*01--Macaca Mulata; nomenclature for rhesus MHC--animals. All animals were inoculated intravenously with ten infectious doses of highly pathogenic SIV251 and, on day 15 after SIV251 infection, sixteen animals (groups A and B) were started on antiretroviral therapy (HAART) with subcutaneous inoculation of 20 mg/kg/day of PMPA, intravenous inoculation of 10 mg/kg/day of Didanosine (DDI), and oral administration of Stavudine (d4T) divided into two doses daily (total of 2.4 mg/kg/day). At weeks 10, 19, and 23 after infection, all macaques in groups B (HAART-treated) and C (untreated) were immunized intramuscularly with 10 to the eighth pfu of NYVAC-SIV-gag-pol-env vaccine, whereas animals in group A (HAART-treated) received 10 to the eighth pfu of mock NYVAC parental virus.In summary, we have achieved the following:1.Establishment of an animal model in which immunization and immune intervention can be assessed in a controlled fashion.2.Demonstration that both CD4+ and CD8+ T-cell responses are elicited/boosted by a vaccine in infected animals treated with HAART. This point is very important in the context of the present concern that HAART may silence the immune response to the detriment of the host.3.The use of this attenuated live vector is safe in animals with active viral replication (group C) as well as in HAART-treated macaques (groups A and B).4.Early intervention with HAART changes the natural course of disease.5.Immunization may have an effect, and we observe a clear trend although, because of the sample size, we were unable to demonstrate the statistical significance of this finding. Because of the encouraging nature of these results, a safety and immunogenicity trial in HIV-I-infected individuals will be initiated the coming year using a NYVAC-based HIV-I vaccine.